Sustained successful peanut oral immunotherapy associated with low basophil activation and peanut-specific IgE

Journal Pre-proof Sustained Successful Peanut Oral Immunotherapy Associated with Low Basophil Activation and Peanut-Specific IgE Mindy Tsai, DMSc, Kaori Mukai, PhD, R. Sharon Chinthrajah, MD, Kari C. Nadeau, MD, PhD, Stephen J. Galli, MD PII:

S0091-6749(19)31620-3

DOI:

https://doi.org/10.1016/j.jaci.2019.10.038

Reference:

YMAI 14285

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 29 July 2019 Revised Date:

27 September 2019

Accepted Date: 31 October 2019

Please cite this article as: Tsai M, Mukai K, Chinthrajah RS, Nadeau KC, Galli SJ, Sustained Successful Peanut Oral Immunotherapy Associated with Low Basophil Activation and Peanut-Specific IgE, Journal of Allergy and Clinical Immunology (2020), doi: https://doi.org/10.1016/j.jaci.2019.10.038. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

Tsai, Mukai et al

Sustained Successful Peanut Oral Immunotherapy Associated with Low Basophil Activation and Peanut-Specific IgE Mindy Tsai, DMSc1,2*, Kaori Mukai, PhD1,2*, R. Sharon Chinthrajah, MD1,2, Kari C. Nadeau, MD, PhD1,2, Stephen J. Galli, MD1,2,3,

1. Department of Pathology; 2. Sean N. Parker Center for Allergy and Asthma Research; 3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA * These authors contributed equally to this work

Correspondence to: Stephen J. Galli, MD Stanford University, 269 Campus Drive, Stanford, CA 94305-5101, USA. E-mail: [email protected] Phone: 650-736-0062 Fax: 540-736-0073

Funding: This work was supported by the National Institutes of Health [grant number U19 AI104209].

Disclosure of potential conflict of interest: No conflict.

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ABSTRACT Background: Oral immunotherapy (OIT) can successfully desensitize many peanut allergic subjects, but clinical tolerance diminishes over time upon discontinuation, or low dose maintenance, of peanut. Therefore, in order to improve the efficacy and sustainability of such therapy, we sought to identify biomarkers and clinical tools that can predict therapeutic outcomes and monitor treatment responses.

Objective: We evaluated whether basophil activation in whole blood, and plasma levels of peanutspecific immunoglobulins, are useful biomarkers for peanut OIT.

Methods: We longitudinally measured, before, during and after OIT, basophil activation in whole blood ex vivo in response to peanut stimulation, and peanut-specific IgE and IgG4, in a large, single-site, double-blind, randomized, placebo-controlled, phase 2 peanut OIT study. We compared basophil responsiveness and peanut specific immunoglobulins between those who were clinically reactive vs. tolerant to peanut oral challenges.

Results: Peanut OIT significantly decreased basophil activation, peanut-specific, Ara h 1, Ara h 2 and Ara h 3 IgEs, and sIgE/total IgE, but increased sIgG4/sIgE. Participants who became reactive to 4 g of peanut 13 weeks off active OIT exhibited higher peanutinduced basophil activation ex vivo and higher peanut-specific IgEs and sIgE/total IgE,

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but lower sIgG4/sIgE. Notably, participants entering the study with low basophil responsiveness were more likely to achieve treatment success. Substantial suppression of basophil activation was required to maintain long-term clinical tolerance after peanut OIT.

Conclusion: Assessments of peanut-specific basophil activation and peanut-specific immunoglobulins can help to predict treatment outcomes, and to differentiate transient desensitization vs. sustained unresponsiveness after OIT.

Key messages: • Peanut OIT suppresses basophil activation in responses to peanut and anti-IgE, reduces peanut-specific, Ara h 1, Ara h 2 and Ara h 3 IgEs, and increases peanutspecific IgG4. • Measurements of basophil activation by peanut and peanut-specific IgEs can help to differentiate those who will achieve transient desensitization vs. sustained tolerance after peanut OIT. • Basophil non/low responders exhibit higher treatment success to peanut OIT and, among other subjects, 80-90% suppression of peanut-specific basophil activation is required to maintain long-term clinical tolerance after peanut OIT.

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Capsule summary: Assessment of basophil activation in whole blood and specific immunoglobulins in plasma before, during and after peanut OIT can help to differentiate transient desensitization vs. sustained unresponsiveness.

Key words: Basophil, basophil activation test, biomarkers, CD63, CD203c, OIT predictors, oral immunotherapy, peanut allergy, peanut-specific IgE, peanut-specific IgG4.

Abbreviations: POISED: Peanut oral immunotherapy study: safety, efficacy and discovery BAT: Basophil activation test OIT: Oral immunotherapy DBPCFC: Double-blind, placebo-controlled, oral food challenge SU: Sustained unresponsiveness ITT: Intention to treat AUC: Area under the curve LR: Basophil non/low responder IR: Basophil intermediate responder HR: Basophil high responder MFI: Mean florescent intensity

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INTRODUCTION There has been an increasing prevalence of food allergy, in both children and adults.1, 2. However, there is currently no cure, and standard care, e.g., dietary avoidance and symptomatic treatments, are suboptimal. Peanut allergy is particularly problematic. Only ~20% of peanut allergic subjects are expected to outgrow this allergy3. Moreover, it is associated with high rates of accidental exposure, which carries a dangerous risk of systematic anaphylaxis4. Finally, diagnosing peanut allergy without oral peanut challenge is clinically challenging5.

Experimental therapies using oral immunotherapy (OIT), sublingual immunotherapy (SLIT), or epicutaneous immunotherapy (EPIT) have evaluated the safety, efficacy, and immunological changes associated with peanut immunotherapy6. Such studies have demonstrated that immunotherapy can successfully desensitize patients to peanut7-16. However, it isn’t clear whether long-term tolerance is sustainable without continuous peanut dosing after active therapy.

We recently completed a single-site, double-blind, randomized, placebo-controlled, phase 2 study (POISED) to evaluate the long-term effects of peanut OIT17. POISED participants underwent peanut OIT build-up and maintenance followed by either peanut withdrawal or low dose (300 mg) peanut daily dosing. More than 80% of participants tolerated up to 4 g of peanut protein after 2 years of active OIT therapy, but the tolerance declined substantially with or without low dose daily peanut intake17. Our findings in POISED17, and others’ studies12, 18-20, indicate that maintenance dosing and

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treatment regimens need to be improved further to achieve long-term protection after peanut immunotherapy.

Biomarkers for improving accuracy in diagnosis, and/or differentiating short-term desensitization vs. long-term unresponsiveness or other outcomes, would be useful in improving treatment protocols that could ultimately be based on an individual’s response to therapy. While several immunological parameters are being evaluated for such purposes, the basophil activation test (BAT) has emerged as one of the most promising tools for food allergy testing21, 22. However, routine applications of BATs in clinical and research settings is not yet feasible due to the skills required to handle blood basophils, the lack of standardization of BAT protocols, and validation of BAT results with clinical outcomes.

To facilitate the utility of BATs, we recently developed a simple BAT protocol that produces consistent and reproducible results using whole blood samples collected in heparin, stored at 4°C, and processed within 24 hou rs after blood collection23. Using this protocol, basophil activation can be quantified in small volumes of whole blood by flow cytometric measurements of activation markers, such as CD63 and CD203c23. In the present study, we performed a comprehensive longitudinal analysis of basophil activation in the whole blood of participants enrolled in the POISED phase 2 peanut OIT study, and examined the association between basophil responsiveness and treatment outcomes after 2 years of peanut OIT followed by 13 weeks of peanut withdrawal (Peanut 0) or 300 mg low dose peanut maintenance (Peanut 300). We reported recently

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that lower peanut IgE and higher sIgG4/sIgE at baseline are associated with treatment success in our POISED study17. In this study, we examined whether OIT-induced changes in peanut specific IgEs and IgG4 are associated with basophil activation and are predictive of sustained outcomes.

METHODS Study design Whole blood for BATs was collected from peanut allergic participants (adults and children) enrolled in the POISED trial (ClinicalTrials.gov NCT02103270) at the Sean N. Parker Center for Allergy and Asthma Research at Stanford University. The clinical research protocol was approved by the Division of Allergy, Immunology, and Transplantation (DAIT)/National Institute of Allergy and Infectious Diseases (NIAID) Allergy and Asthma Data Safety Management Board, the DAIT/NIAID Clinical Review Committee, the Stanford Institutional Review Board, and the Food and Drug Administration (FDA). For complete study design, baseline characteristics of the participants, and treatment outcomes, see Chinthrajah et al17. Briefly, 120 participants who reacted to a cumulative ingested dose of < 500 mg peanut by DBPCFCs at enrollment were randomized into three arms: Peanut 0 (60 participants), Peanut 300 (35 participants) and Placebo (25 participants). Peanut 0 and Peanut 300 participants received OIT build up to, and maintenance of, 4 g of peanut protein until week 104, when participants in the Peanut 0 arm discontinued peanut and those in the Peanut 300 arm maintained daily dosing of 300 mg peanut protein. Placebo received oat flour throughout the study. DBPCFCs were performed at baseline (week 0), week 104, week

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117, and every 13 weeks for those who passed the previous DBPCFCs until the last DBPCFC at week 156. Intention-to- treat (ITT) populations included all 120 participants randomized at enrollment. Treatment “success” is defined as passing DBPCFCs to 4 g of peanut protein. Participants exiting the study before week 104 were considered “dropouts”. There were 9 dropouts in Peanut 0, 5 dropouts in Peanut 300, and 3 dropouts in Placebo, including 2 Placebo participants who exited the clinical study before week 104 and another Placebo participant whose basophil data were not available at weeks 104 and 117. All dropouts were excluded from longitudinal analyses. One participant was randomized to Peanut 300, failed DBPCFC at week 104, and was not on 300 mg peanut from weeks 104-117, nor received food challenge at week 117. Data from this participant were included up to week 104.

Blood collection Blood was collected from each participant before, during, and after OIT at weeks 0, 12, 52, 104 and 117.

Basophil activation test BATs were performed using whole blood preserved in heparin as described in Mukai et al23. Briefly, basophil activation was assessed after stimulation for 30 min at 37°C with IL-3 (PeproTech, 2 ng/mL), polyclonal rabbit anti-human IgE (Bethyl Laboratoies, 1 µg/mL), or peanut protein extracted from the same peanut flour used in OIT at 0, 0.1, 1, 10, 100, 1000 (ng/mL). All stimuli were prepared in RPMI. Basophils were gated as CD123+HLA-DR- cells and %CD63high basophils and CD203c expression levels were

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quantified by flow cytometry. To assess changes in basophil reactivity and sensitivity in responses to OIT, up-regulation of the basophil activation marker CD63 induced by peanut ex vivo stimulation was calculated as area under curve (AUC) of %CD63high basophils as a function of the peanut dose24. %CD63high PE AUC was calculated for each participant at each time point. Expression of CD203c was measured by MFI. ∆CD203c is defined as CD203c MFI after anti-IgE, IL-3, or peanut stimulation minus CD203c MFI incubated with RPMI.

Immunoglobulin measurements Total IgE, peanut-specific IgE and IgG4 levels and component IgEs (Ara h 1, 2, 3) were measured at baseline, week 104 and week 117 using standardized methods in CLIAapproved laboratories (Johns Hopkins University for peanut-specific IgE, peanutspecific IgG4, and component IgE testing, and Stanford Clinical laboratories for total IgE measurements). Both laboratories (at Stanford and Johns Hopkins) measured immunoglobulin levels using ImmunoCAP on the Phadia 250 Immunoassay Analyzers (Thermo Fisher Scientific).

Statistical analysis Statistical comparisons were performed using GraphPad Prism 8 or R version 3.6.0. Mixed-effects analysis with the Geisser-Greenhouse correction was used for the longitudinal analysis of basophil CD63 and CD203c. Mann-Whitney tests were performed for comparisons between different treatment arms and between success and failure outcomes. Wilcoxon matched-pairs signed rank test was used to assess changes

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in immunoglobulin levels in the same group. Spearman correlations were used to assess associations between basophil activation and immunoglobulins levels. Fisher’s exact test was used to test for differences in treatment outcomes between basophil responders.

RESULTS Peanut OIT suppresses basophil activation and reduces peanut specific IgE Before OIT, there were no differences in the induction of %CD63high basophils by ex vivo stimulation with peanut or anti-IgE between Peanut 0, Peanut 300 and Placebo (Fig E1. A, B). Levels of CD203c expression in basophils after anti-IgE, IL-3 or peanut stimulation were also similar between the 3 treatment arms at baseline (Fig E1. C). Consistent with previous OIT studies of peanut allergy7, 18, 25-27, our OIT protocol significantly decreased peanut-induced %CD63high basophils as early as week 12 of the OIT build up phase (Fig 1, A). Up-regulation of CD63 in basophils was suppressed during the OIT maintenance phase from weeks 52-104. Basophils of Peanut 0 and Peanut 300 participants continued to exhibit reduced levels of peanut induced activation until week 117 (Fig 1, A).

Peanut OIT also significantly reduced %CD63high basophils in response to anti-IgE (1 µg/mL) stimulation in Peanut 0 and Peanut 300 participants as early as week 12 (Fig 1, B). Interestingly, we detected a small but significant increase in peanut-, but not antiIgE-, induced %CD63high basophils in Peanut 0 participants between week 104 and week 117. This increase is likely due to peanut avoidance by the Peanut 0 participants

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during this period. On the other hand, there was no significant difference in %CD63high PE AUC between weeks 104 and 117 in Peanut 300, during which these participants continued taking 300 mg peanut after active OIT (Fig 1).

Similarly, peanut OIT significantly inhibited basophil CD203c expression induced by anti-IgE, IL-3 and peanut stimulation over time (Fig E2, A, B). No significant change in CD63 and substantially lesser changes in CD203c were observed in basophils of Placebo participants (Fig 1 and Fig E2, C). Basophil responses were strongly suppressed by peanut OIT at weeks 104 and 117 compared to Placebo, however, there was no difference in peanut-induced basophil activation between Peanut 0 and Peanut 300 participants (Fig E3) at either time, suggesting 300 mg peanut maintenance did not influence basophil responses at this timepoint.

Our peanut OIT also significantly decreased plasma levels of peanut, Ara h 1, Ara h 2 and Ara h 3 IgEs, and the ratio of peanut specific IgE (sIgE) vs. total IgE (Fig 2). By contrast, peanut specific IgG4 (sIgG4), and ratios of sIgG4 vs. sIgE were elevated by peanut OIT (Fig 2). There were no changes in sIgE, Ara h 1, Ara h 2, or Ara h 3 IgE, sIgE/total IgE or sIgG4/sIgE between week 0 and 117 in Placebo participants (Table E1). However, we detected some small but significant reductions in IgEs specific for peanut, Ara h 1 and Ara h 2, and sIgE/total IgE, in Placebo participants at week 104. Importantly, IgEs specific for peanut, Ara h 1, Ara h 2 and Ara h 3, and sIgG4, sIgE/total IgE, and sIgG4/sIgE, between Peanut 0 and Peanut 300 participants were not different

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at week 117, indicating that 300 mg of peanut dosing for an additional 13 weeks after active OIT did not influence plasma levels of peanut specific immunoglobulins.

Correlations of basophil activation and peanut specific immunoglobulins Induction of allergen-specific IgG4 is a common outcome of immunotherapy and thought to be the primary mechanism that inhibits IgE-mediated effector cell activation28, 29

. We recently reported lower peanut-induced basophil activation and higher peanut

sIgG4/sIgE before treatment is associated with OIT-induced sustained effects (i.e. passing DBPCFCs to 4 g peanut) after 13 weeks of peanut avoidance17. Furthermore, lower Ara h 2 IgE and peanut specific IgE are significantly associated with treatment success at primary end point at week 117 in both Peanut 0 and Peanut 300 participants, and baseline sIgE/total IgE is associated with a higher % of dose-related adverse events17.

We examined whether basophil activation was correlated with the plasma levels of sIgE or sIgG4 before and after active OIT treatment. At baseline before OIT, there were weak, but significant correlations between peanut-induced basophil activation (%CD63high PE AUC) vs. IgEs specific for peanut, Ara h 1, Ara h 2 or Ara h 3, or sIgE/total IgE, in 120 intention-to-treat (ITT) participants (Fig 3, A). While there was no correlation between basophil responsiveness and sIgG4, we detected a weak, but significant, negative correlation between %CD63high PE AUC and sIgG4/sIgE at baseline (Fig 3, A).

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We analyzed the primary end-point at week 117 (i.e., 13 weeks after active OIT treatment). We found significant weak to moderate correlations between peanutinduced basophil activation and IgE for peanut, Ara h 1, Ara h 2 or Ara h 3, and sIgE/total IgE or sIgG4/sIgE, but no correlation between sIgG4 and peanut-induced basophil activation (Fig 3, B). These data suggest that peanut-induced basophil activation is regulated not just by specific IgG4 alone, but by the interactions of basophils with peanut specific IgEs, peanut specific IgG4, and total IgE.

Peanut-induced basophil activation and specific IgEs differentiate transient desensitization vs. sustained unresponsiveness The main goals of our peanut OIT study were to: 1) assess sustained unresponsiveness to a cumulative dose of 4 g peanut protein after active peanut OIT treatment followed by 13 weeks of peanut avoidance, and 2) test the sustained effect of 300 mg daily dosing following active therapy. We compared longitudinally, between those passing vs. failing food challenges at week 117, peanut-induced %CD63high basophils at weeks 0, 12, 52, 104 and 117.

Peanut 0 and Peanut 300 participants who failed 4 g peanut DBPCFCs at week 117 exhibited higher %CD63high basophils upon peanut stimulation at every time (Fig 4, A, B), while there was no significant difference in %CD63high basophils to anti-IgE stimulation (1 µg/mL) between those who passed vs. failed week 117 peanut challenges (Fig 4, C, D). Peanut 0 participants who failed week 117 oral food challenge also exhibited higher ∆CD203c in response to peanut stimulation, but not to anti-IgE or IL-3

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(Fig E4, A, B). In Peanut 300, lower ∆CD203c in basophils of those who passed 4 g peanut DBPCFCs at week 117 were detected with high dose peanut stimulation at week 117 (Fig E4, C, D). In addition, participants who failed week 117 DBPCFCs exhibited significantly higher IgEs specific for peanut, Ara h 1, Ara h 2 and Ara h 3, and sIgE/total IgE, at week 117 (Fig 5). Peanut 0 participants who failed week 117 DBPCFCs had (unexpectedly) significantly higher sIgG4, but lower sIgG4/sIgE, at week 117 (Fig 5, A). sIgG4 and sIgG4/sIgE were not different between Peanut 300 participants who failed vs. passed week 117 DBPCFCs (Fig 5, B).

We did not find any significant differences between Peanut 0 and Peanut 300 participants who failed or between Peanut 0 and Peanut 300 participants who passed week 117 DBPCFCs in %CD63high PE AUC at weeks 0, 12, 52, 104 and 117, or in ∆CD203c and peanut specific IgEs and IgG4 at week 0 and week 117.

Basophil non/low responders to peanut exhibit lower peanut-specific IgE and better treatment outcome Basophils of the 120 participants enrolled in this study expressed a wide range of peanut-induced CD63 upregulation before treatment. Based on the sensitivity and extent of their basophil responses to ex vivo peanut stimulation at enrollment, participants could be classified as basophil “non/low”, “intermediate”, or “high’ responders. We found that the induction of %CD63high basophils (calculated by AUC as a function of the peanut dose response curves [0-1000 ng/mL]) ranged from 0 to 192.6 with a mean of 54.73 (Fig 6, A). We defined the “non/low responders” (LR) as those with

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peanut-induced %CD63high dose response AUC<12.09 (= mean (54.73)-1x SD (42.64) of %CD63high PE AUC of 120 participants) and the “high responders” (HR) as those with peanut induced %CD63high dose response AUC>97.37 (= mean+1xSD of %CD63high PE AUC of 120 participants). The “intermediate responders” (IR) had peanut-induced %CD63high dose response AUC >12.09 and <97.37 (Fig 6, B).

There were 18 (15%) LRs, 83 (69%) IRs and 19 (16%) HRs in our ITT population (Fig 6, C). Moreover, there was a similar distribution of LRs, IRs and HRs among the three treatment arms (Figure 6, C). Participants were classified based on their basophil responses to peanut, but we found that the basophil LRs to peanut also exhibited lower anti-IgE induced CD63 up-regulation and vice versa for the HRs (Fig 6, D). On the other hand, we found that basophil LRs tolerated 2-3 times more peanut protein at enrollment than the IRs and HRs (LR vs. IR vs HR: [150.6 + 35.51] mg vs. [66.51+ 10.92] mg vs. [49.21+ 15.96] mg) (Fig 6, E). Furthermore, before treatment, basophil LRs also had lower peanut-specific, Ara h 1, Ara h 2 and Ara h 3 IgE, and sIgE/total IgE, compared to IRs and/or HRs (Fig 7, A). By contrast, basophil HRs had higher levels of peanutspecific, Ara h 1, Ara h 2 and Ara h 3 IgE, and sIgE/total IgE. Interestingly, no difference was detected in levels of peanut specific IgG4 among LRs, IRs or HRs, but the ratio of sIgG4/sIgE was significantly higher in LRs compared to IRs or HRs (Fig 7, A).

We also compared plasma IgE and IgG4 levels between the 3 groups of basophil responders at week 117 after active OIT treatment. Basophil LRs exhibited the lowest levels of IgE specific for peanut, Ara h 1, Ara h 2 and Ara h 3 compared to IRs and/or

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HRs, whereas the levels of these IgEs were not different between IRs and HRs (Fig 7, B). Notably, the levels of peanut-specific IgG4 were also lower in the basophil LRs compared to either IRs or HRs after OIT, whereas peanut sIgG4/sIgE was not significantly different between 3 groups after OIT (Fig 7, B).

LRs not only exhibited lower basophil activation following peanut or anti-IgE stimulation, and lower peanut specific IgEs, they also responded to OIT with better outcomes. 91% and 55% of LRs passed oral food challenge up to 4 g of peanut protein at week 117 and week 156, respectively (Table 1). By contrast, only 17% of basophil high responders passed oral food challenges at both weeks 117 and 156. 48% and 22% of the IR recorded treatment successes at weeks 117 and 156, respectively.

Notably, levels of peanut-induced %CD63high basophils of IRs and HRs in the Peanut 0 arm who passed week 117 DBPCFCs were reduced to 20 + 4% of their baseline levels by OIT, whereas those who failed still had 42 + 7% of their baseline levels (P = 0.01). Similarly, peanut-induced %CD63high basophils in IRs and HRs in the Peanut 300 arm who passed week 117 DBPCFCs were reduced to 11 + 2% of their baseline levels, compared to 45 + 18% in those who failed (P < 0.0001).

These data indicate that achieving sustained unresponsiveness 13 weeks off active OIT is observed primarily in two groups of subjects, those with lower basophil responses at study entrance and those that undergo a substantial reduction (80-90%) of their peanutinduced basophil activation after OIT.

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Basophil responsiveness in LR participants who reached treatment success remained low after OIT (%CD63high PE AUC=3.78 + 1.21 & 5.67 + 2.45 at week 0 & week 117, respectively. P = 0.30). Peanut OIT also did not alter specific IgE in these LRs (peanut specific IgE (kU/L) =25.70 + 10.92 & 13.39 + 5.26 at week 0 & week 117, respectively. P = 0.13), but increased specific IgG4 (ng/mL) levels from 0.82 + 0.30 at week 0 to 8.93 + 4.53 at week 117 (P = 0.03). By contrast, sustained tolerance in IR and HR subgroups was associated with lower %CD63high PE AUC and lower IgEs specific for peanut, Ara h 1, Ara h 2 and Ara h 3, but not with higher sIgG4, before and/or after OIT treatment (Table E2).

In summary, in our POISED study, assessment of basophil activation and measurement of peanut specific IgEs are useful biomarkers in differentiating transient desensitization vs. sustained tolerance.

DISCUSSION Since its first application in food allergy two decades ago30, the BAT has been actively evaluated in this disorder21, 22. Enhanced spontaneous and food-induced basophil activation was shown to be associated with clinical reactivity to milk31, 32, peanut33, 34, egg33, 35, red meat36 and other foods21, 22. Some studies have suggested that BATs are more reliable than skin prick tests and specific IgE levels in differentiating food allergy from tolerance in children32, 34. While suppression of basophil activation by immunotherapy has been demonstrated in food allergy to egg37, milk38, and peanut7, 8, 18, 25-27, 39, 40

, only a few studies have examined the association of basophil activation

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with treatment outcomes18, 26, 27, 37, 40, 41. Our analysis of BAT results obtained for more than 2 years in the POISED study shows that basophil activation remains suppressed even after 13 weeks off active OIT, and that sustained protection is associated with lower levels of basophil activation by peanut before, during, and after OIT.

In addition to inhibiting peanut-induced basophil activation, our peanut OIT also decreased upregulation of CD63 and CD203c in responses to anti-IgE and/or IL-3. Antigen-nonspecific inhibition of basophil activation also was observed in other peanut OIT studies18, 25. This “bystander effect” is likely due to reduction of FcεRI on basophils as a consequence of IgE reduction by OIT42, 43.

The mechanisms by which OIT suppress basophil activation are still being investigated. We think that understanding the suppression of basophils by immunotherapy can help in efforts to devise better treatment strategies or preventive approaches for allergies to peanuts and other foods. Depletion of IgG, specifically IgG4, has been shown to eliminate the inhibitory effect of OIT plasma in basophil activation28, 40, 44. We did not find direct associations between peanut induced %CD63high basophils and levels of peanut specific IgG4 alone, however, we detected significant weak correlations between peanut-induced %CD63high basophils with sIgG4/sIgE before and after OIT, as well as a moderate correlation between peanut-induced %CD63high basophils and sIgE/total IgE after OIT. These findings suggest that basophil activation is regulated by the summation of activating (such as sIgE) and inhibitory (e.g., IgG1, IgG4) immunoglobulins. Indeed,

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our findings indicate not only that the levels of sensitization at study entrance, but also immune responses shaped by OIT, can have significant impacts on treatment outcome.

Furthermore, other immunoglobulin isotypes induced by OIT can also potentially inhibit basophil activation45, 46. It is conceivable that peanut immunotherapy induces immunological modifications (such as reduction of peanut specific IgE and increase of peanut specific IgG, IgA, and IgD) which in turn alter basophil phenotype (such as reduction of FcεR, Syk47 and other intracellular signaling machinery) and thus reduces basophil responsiveness to food allergens, and clinical reactivity. Therefore, we think that BATs ideally should be performed in whole blood, as we did in this study, to evaluate OIT-induced immune modifications external to basophils, as well as intrinsic changes in basophils.

We found that participants with or without 300 mg peanut daily dosing after OIT exhibited similar basophil responsiveness and peanut-specific immunoglobulins tested 13 weeks after active OIT. These findings are in line with clinical observations that Peanut 0 and Peanut 300 participants achieved similar treatment success rates 13 weeks after active OIT17. However, Peanut 300 participants were 3 times more likely to tolerate 4 g peanut DBPCFCs than Peanut 0 participants one year after active OIT. One limitation of our study is that we were not able to associate BAT results with clinical outcomes beyond 3 months after stopping active OIT, because most of the participants who failed week 117 DBPCFCs did not return to the study and their basophils were not tested further.

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In summary, we have demonstrated that BATs and blood biomarkers (including peanut and peanut component-specific IgEs, sIgG4/sIgE, and sIgE/total IgE) measured before, during, and after immunotherapy, can differentiate transient desensitization vs. sustained tolerance after peanut OIT. Our analysis of basophil responsiveness to ex vivo peanut stimulation reveals phenotypic heterogeneity among basophils in peanut allergy, and identifies a peanut allergy subpopulation (i.e., basophil non/low responders) who are more amenable to OIT treatment. Our results support the hypothesis that BATs, in conjunction with other blood biomarkers, can identify allergic subjects who can most benefit from OIT. They also raise the possibility that prolonged administration of antigen, or antigen updosing, may improve the efficacy and durability of OIT in difficultto-treat populations.

ACKNOWLEDGMENTS This project was supported by the NIAID AADCRC U19 AI104209. We thank Dr. Chen Liu for technical assistance, Ms. Shu Cao, Ms. Natasha Purington and Dr. Rob Tibshirani for statistical consultation, and all of the participants of the POISED study for their cooperation.

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In: Oria MP, Stallings VA, editors. Finding a Path to Safety in Food Allergy: Assessment of the Global Burden, Causes, Prevention, Management, and Public Policy. Washington (DC); 2016. Renz H, Allen KJ, Sicherer SH, Sampson HA, Lack G, Beyer K, et al. Food allergy. Nat Rev Dis Primers 2018; 4:17098. Skolnick HS, Conover-Walker MK, Koerner CB, Sampson HA, Burks W, Wood RA. The natural history of peanut allergy. J Allergy Clin Immunol 2001; 107:367-74. Cherkaoui S, Ben-Shoshan M, Alizadehfar R, Asai Y, Chan E, Cheuk S, et al. Accidental exposures to peanut in a large cohort of Canadian children with peanut allergy. Clin Transl Allergy 2015; 5:16. Koplin JJ, Perrett KP, Sampson HA. Diagnosing Peanut Allergy with Fewer Oral Food Challenges. J Allergy Clin Immunol Pract 2019; 7:375-80. Vickery BP, Ebisawa M, Shreffler WG, Wood RA. Current and Future Treatment of Peanut Allergy. J Allergy Clin Immunol Pract 2019; 7:357-65. Jones SM, Pons L, Roberts JL, Scurlock AM, Perry TT, Kulis M, et al. Clinical efficacy and immune regulation with peanut oral immunotherapy. J Allergy Clin Immunol 2009; 124:292-300, e1-97. Kim EH, Bird JA, Kulis M, Laubach S, Pons L, Shreffler W, et al. Sublingual immunotherapy for peanut allergy: clinical and immunologic evidence of desensitization. J Allergy Clin Immunol 2011; 127:640-6 e1. Varshney P, Jones SM, Scurlock AM, Perry TT, Kemper A, Steele P, et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol 2011; 127:654-60. Fleischer DM, Burks AW, Vickery BP, Scurlock AM, Wood RA, Jones SM, et al. Sublingual immunotherapy for peanut allergy: a randomized, double-blind, placebocontrolled multicenter trial. J Allergy Clin Immunol 2013; 131:119-27 e1-7. Anagnostou K, Islam S, King Y, Foley L, Pasea L, Bond S, et al. Assessing the efficacy of oral immunotherapy for the desensitisation of peanut allergy in children (STOP II): a phase 2 randomised controlled trial. Lancet 2014; 383:1297-304. Narisety SD, Frischmeyer-Guerrerio PA, Keet CA, Gorelik M, Schroeder J, Hamilton RG, et al. A randomized, double-blind, placebo-controlled pilot study of sublingual versus oral immunotherapy for the treatment of peanut allergy. J Allergy Clin Immunol 2015; 135:1275-82 e1-6. Vickery BP, Berglund JP, Burk CM, Fine JP, Kim EH, Kim JI, et al. Early oral immunotherapy in peanut-allergic preschool children is safe and highly effective. J Allergy Clin Immunol 2017; 139:173-81 e8. Bird JA, Spergel JM, Jones SM, Rachid R, Assa'ad AH, Wang J, et al. Efficacy and Safety of AR101 in Oral Immunotherapy for Peanut Allergy: Results of ARC001, a Randomized, Double-Blind, Placebo-Controlled Phase 2 Clinical Trial. J Allergy Clin Immunol Pract 2018; 6:476-85 e3. Investigators PGoC, Vickery BP, Vereda A, Casale TB, Beyer K, du Toit G, et al. AR101 Oral Immunotherapy for Peanut Allergy. N Engl J Med 2018; 379:1991-2001. Blumchen K, Trendelenburg V, Ahrens F, Gruebl A, Hamelmann E, Hansen G, et al. Efficacy, Safety, and Quality of Life in a Multicenter, Randomized, Placebo-Controlled Trial of Low-Dose Peanut Oral Immunotherapy in Children with Peanut Allergy. J Allergy Clin Immunol Pract 2019; 7:479-91 e10. Chinthrajah RS, Purington N, Andorf S, Long A, O'Laughlin KL, Lyu SC, et al. Sustained Outcomes in a Large Double-blind, Placebo-controlled, Randomized Phase 2 Study of

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Peanut Immunotherapy. Lancet 2019:Sep 12. pii: S0140-6736(19)31793-3. doi: 10.1016/S0140-6736(19)-3. [Epub ahead of print]. Gorelik M, Narisety SD, Guerrerio AL, Chichester KL, Keet CA, Bieneman AP, et al. Suppression of the immunologic response to peanut during immunotherapy is often transient. J Allergy Clin Immunol 2015; 135:1283-92. Chu DK, Wood RA, French S, Fiocchi A, Jordana M, Waserman S, et al. Oral immunotherapy for peanut allergy (PACE): a systematic review and meta-analysis of efficacy and safety. Lancet 2019; 393:2222-32. Cook QS, Kim EH. Update on peanut allergy: Prevention and immunotherapy. Allergy Asthma Proc 2019; 40:14-20. Santos AF, Lack G. Basophil activation test: food challenge in a test tube or specialist research tool? Clin Transl Allergy 2016; 6:10. Santos AF, Shreffler WG. Road map for the clinical application of the basophil activation test in food allergy. Clin Exp Allergy 2017; 47:1115-24. Mukai K, Gaudenzio N, Gupta S, Vivanco N, Bendall SC, Maecker HT, et al. Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis. J Allergy Clin Immunol 2017; 139:889-99 e11. Hoffmann HJ, Santos AF, Mayorga C, Nopp A, Eberlein B, Ferrer M, et al. The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease. Allergy 2015; 70:1393-405. Thyagarajan A, Jones SM, Calatroni A, Pons L, Kulis M, Woo CS, et al. Evidence of pathway-specific basophil anergy induced by peanut oral immunotherapy in peanutallergic children. Clin Exp Allergy 2012; 42:1197-205. Chin SJ, Vickery BP, Kulis MD, Kim EH, Varshney P, Steele P, et al. Sublingual versus oral immunotherapy for peanut-allergic children: a retrospective comparison. J Allergy Clin Immunol 2013; 132:476-8 e2. Kulis M, Yue X, Guo R, Zhang H, Orgel K, Ye P, et al. High- and low-dose oral immunotherapy similarly suppress pro-allergic cytokines and basophil activation in young children. Clin Exp Allergy 2019; 49:180-9. Santos AF, James LK, Bahnson HT, Shamji MH, Couto-Francisco NC, Islam S, et al. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens. J Allergy Clin Immunol 2015; 135:1249-56. Kulis MD, Patil SU, Wambre E, Vickery BP. Immune mechanisms of oral immunotherapy. J Allergy Clin Immunol 2018; 141:491-8. Moneret-Vautrin DA, Sainte-Laudy J, Kanny G, Fremont S. Human basophil activation measured by CD63 expression and LTC4 release in IgE-mediated food allergy. Ann Allergy Asthma Immunol 1999; 82:33-40. Wanich N, Nowak-Wegrzyn A, Sampson HA, Shreffler WG. Allergen-specific basophil suppression associated with clinical tolerance in patients with milk allergy. J Allergy Clin Immunol 2009; 123:789-94 e20. Rubio A, Vivinus-Nebot M, Bourrier T, Saggio B, Albertini M, Bernard A. Benefit of the basophil activation test in deciding when to reintroduce cow's milk in allergic children. Allergy 2011; 66:92-100. Ocmant A, Mulier S, Hanssens L, Goldman M, Casimir G, Mascart F, et al. Basophil activation tests for the diagnosis of food allergy in children. Clin Exp Allergy 2009; 39:1234-45. Santos AF, Douiri A, Becares N, Wu SY, Stephens A, Radulovic S, et al. Basophil activation test discriminates between allergy and tolerance in peanut-sensitized children. J Allergy Clin Immunol 2014; 134:645-52.

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Berin MC, Grishin A, Masilamani M, Leung DYM, Sicherer SH, Jones SM, et al. Eggspecific IgE and basophil activation but not egg-specific T-cell counts correlate with phenotypes of clinical egg allergy. J Allergy Clin Immunol 2018. Mehlich J, Fischer J, Hilger C, Swiontek K, Morisset M, Codreanu-Morel F, et al. The basophil activation test differentiates between patients with alpha-gal syndrome and asymptomatic alpha-gal sensitization. J Allergy Clin Immunol 2019; 143:182-9. Burks AW, Jones SM, Wood RA, Fleischer DM, Sicherer SH, Lindblad RW, et al. Oral immunotherapy for treatment of egg allergy in children. N Engl J Med 2012; 367:233-43. Keet CA, Frischmeyer-Guerrerio PA, Thyagarajan A, Schroeder JT, Hamilton RG, Boden S, et al. The safety and efficacy of sublingual and oral immunotherapy for milk allergy. J Allergy Clin Immunol 2012; 129:448-55, 55 e1-5. Syed A, Garcia MA, Lyu SC, Bucayu R, Kohli A, Ishida S, et al. Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3). J Allergy Clin Immunol 2014; 133:500-10. Orgel K, Burk C, Smeekens J, Suber J, Hardy L, Guo R, et al. Blocking antibodies induced by peanut oral and sublingual immunotherapy suppress basophil activation and are associated with sustained unresponsiveness. Clin Exp Allergy 2019; 49:461-70. Patil SU, Steinbrecher J, Calatroni A, Smith N, Ma A, Ruiter B, et al. Early decrease in basophil sensitivity to Ara h 2 precedes sustained unresponsiveness after peanut oral immunotherapy. J Allergy Clin Immunol 2019:Aug 1. pii: S0091-6749(19)30977-7. doi: 10.1016/j.jaci.2019.07.028. [Epub ahead of print]. Malveaux FJ, Conroy MC, Adkinson NF, Jr., Lichtenstein LM. IgE receptors on human basophils. Relationship to serum IgE concentration. J Clin Invest 1978; 62:176-81. MacGlashan D, Jr., McKenzie-White J, Chichester K, Bochner BS, Davis FM, Schroeder JT, et al. In vitro regulation of FcepsilonRIalpha expression on human basophils by IgE antibody. Blood 1998; 91:1633-43. Burton OT, Logsdon SL, Zhou JS, Medina-Tamayo J, Abdel-Gadir A, Noval Rivas M, et al. Oral immunotherapy induces IgG antibodies that act through FcgammaRIIb to suppress IgE-mediated hypersensitivity. J Allergy Clin Immunol 2014; 134:1310-7 e6. Wright BL, Kulis M, Orgel KA, Burks AW, Dawson P, Henning AK, et al. Componentresolved analysis of IgA, IgE, and IgG4 during egg OIT identifies markers associated with sustained unresponsiveness. Allergy 2016; 71:1552-60. Shan M, Carrillo J, Yeste A, Gutzeit C, Segura-Garzon D, Walland AC, et al. Secreted IgD Amplifies Humoral T Helper 2 Cell Responses by Binding Basophils via Galectin-9 and CD44. Immunity 2018; 49:709-24 e8. Macglashan D, Miura K. Loss of syk kinase during IgE-mediated stimulation of human basophils. J Allergy Clin Immunol 2004; 114:1317-24.

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Table 1. Low basophil activation at week 0 is associated with sustained effectiveness of peanut OIT.

Basophil status (week 0) Non/low responder Intermediate responder High responder

Peanut 0+Peanut 300 Wk 117 DBPCFC Wk 156 DBPCFC* Pass Fail P Pass Fail P value value

Peanut 0 Wk 117 DBPCFC Wk 156 DBPCFC* Pass Fail P Pass Fail P value value

10 (91%) 28 (48%) 2 (17%)

5 (83%) 15 (42%) 1 (11%)

1 (9%) 30 (52%) 10 (83%)

0.001

6 (55%) 13 (22%) 2 (17%)

5 (45%) 45 (78%) 10 (83%)

0.08

1 (27%) 21 (58%) 8 (89%)

0.02

2 (33%) 5 (14%) 1 (11%)

4 (67%) 31 (86%) 8 (89%)

0.48

Peanut 300 Wk 117 DBPCFC Wk 156 DBPCFC* Pass Fail P Pass Fail P value value 5 (100%) 13 (59%) 1 (33%)

0 (0%) 9 (41%) 2 (67%)

0.13

4 (80%) 8 (36%) 1 (33%)

1 (20%) 14 (64%) 2 (67%)

0.23

P values were determined by Fisher’s exact test. * Those who failed at week 117 were not retested (for DBPCFC) at week 156. However, in the table, these participants are included as “failures”.

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FIGURE LEGENDS FIG 1. Peanut OIT reduces basophil responsiveness to peanut and anti-IgE over time. A. Peanut-induced %CD63high basophils represented by area under the curve (AUC) as a function of the peanut extract (PE) protein dose (0-1000 ng/mL) in Peanut 0, Peanut 300, and Placebo arms at weeks 0, 12, 52, 104 and 117. B. Anti-IgE (1 µg/mL) induced %CD63high basophils in Peanut 0, Peanut 300 and Placebo arms at weeks 0, 12, 52, 104 and 117. Data from dropout participants (i.e., those who exited the study before week 104) were excluded. Whiskers represent min to max, boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. Individual values are shown as circles. P values were determined by mixed-effects analysis with the Geisser-Greenhouse correction for longitudinal changes, or by Wilcoxon matchedpairs signed rank test for comparisons between adjacent columns. Source of Wk 0 data in A were from Fig 4 of Chinthrajah et al (Ref. 17) with permission from the Publisher (ELSEVIER).

FIG 2. Changes in peanut-specific immunoglobulins associated with peanut OIT. Plasma immunoglobulins were quantified at weeks 0, 104 and 117. A. Peanut 0. B. Peanut 300. Values <0.1 (equivalent to zero) in Ara h 1, h 2 or h 3 IgEs are displayed as 0.01. P values were determined by the Wilcoxon matched-pairs signed rank test. * P<0.05, **P<0.01, ***P<0.001. N.S. P>0.05. Horizontal bar = median. Data from dropout participants (i.e. those who exited the study before week 104) were excluded.

FIG 3. Correlations between basophil responses (%CD63high PE AUC) and plasma

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immunoglobulins before and after OIT. A. Baseline at week 0. All participants in ITT were included in the analysis. B. 13 weeks after active OIT at week 117. Data from dropout participants (i.e., those who exited the study before week 104) were excluded. Values <0.1 in Ara h 1, h 2 and h 3 IgEs are displayed as 0.01. Spearman correlation coefficient (r) and P values are provided on each graph. In B, dots in red circles are data from 2 placebo participants and one basophil high responder in the Peanut 0 arm who failed week 117 DBPCFC.

FIG 4. Peanut- and anti-IgE-induced %CD63high basophils over time by treatment outcome at week 117. A. Peanut-induced %CD63high basophils are represented by area under the curve (AUC) as a function of the peanut extract (PE) protein dose (01000 ng/mL) in Success vs. Failure in Peanut 0. B. Peanut-induced %CD63high basophils are represented by area under the curve (AUC) as a function of the peanut extract (PE) protein dose (0-1000 ng/mL) in Success vs. Failure in Peanut 300. C. AntiIgE (1 µg/mL) induced %CD63high basophils in in Success vs. Failure in Peanut 0. D. Anti-IgE (1 µg/mL) induced %CD63high basophils in Success vs. Failure in Peanut 300. Whiskers represent min to max, boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. Individual values are shown as circles. P values were determined by Mann-Whitney test. “Success” is tolerant to DBPCFCs to 4 g peanut protein at week 117.

FIG 5. Plasma immunoglobulin levels by treatment outcome at week 117. Plasma immunoglobulins were quantified at week 117. A. Peanut 0. B. Peanut 300. Horizon bar

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= median. Values <0.1 in Ara h 1, h 2 and h 3 IgEs are displayed as 0.01. P values were determined by Mann-Whitney test. “Success” is tolerant to DBPCFCs to 4 g peanut protein at week 117. Source of the data in this figure is from Figs S5 and S6 of Chinthrajah et al (Ref. 17) with permission from the Publisher (ELSEVIER).

FIG 6. Three subgroups of peanut allergy participants based on basophil responses to peanut before OIT. A. Peanut induced %CD63high basophils (%CD63high PE AUC) in the 120 participants at baseline. Scatter dot plot with mean + SD. B. Peanut-induced %CD63high basophils of non/low responders (PE AUC<12.09), intermediate responders (PE AUC >12.09 and <97.37), and high responders (PE AUC>97.37). Individual values are shown as circles. C. Distribution of three subgroups of basophil responders in intention-to-treat (ITT) population, Peanut 0, Peanut 300 and Placebo. D. Anti-IgE (1 µg/mL) induced %CD63high basophils in the three subgroups of basophil responders. Whiskers represent min to max, boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. Individual values are shown as circles. E. Cumulative tolerated peanut challenge dose (CTD) at week 0 among the three subgroups of basophil responders. Data are mean+ SEM. LR, non/low responder; IR, intermediate responder, HR, high responder. P values were determined by Mann-Whitney test.

FIG. 7. Plasma immunoglobulin levels in the three groups of basophil responders before and after OIT. A. Before OIT at week 0. Data from ITT participants were included in the analysis. B. After OIT at week 117. Data from Peanut 0 and Peanut 300

27

Tsai, Mukai et al

participants were included; data from dropouts were excluded. Horizon bar = median. LR, non/low responder; IR, intermediate responder; HR, high responder. Values <0.1 in Ara h 1, h 2 and h 3 IgEs were displayed as 0.01. P values were determined by MannWhitney test.

28

Figure 1 Peanut 0

Peanut 300

Placebo

A Mixed effects analysis: P<0.0001

*** ***

B

***

*

Mixed effects analysis: P<0.0001

***

*

**

Mixed effects analysis: P<0.0001

NS

***

**

NS

Mixed effects analysis: P=0.01

Mixed effects analysis: P=0.11

NS

NS

NS

NS

Mixed effects analysis: P=0.22

NS NS

NS

**

NS

NS

NS

*

NS

Wk0 vs 104 vs 117-A

101 100 10-1

102 101 100 10-1 10-2

***

***

*

100 10-1 10-2

10-4

Wk0

Wk104

*

101 100 10-1

102 101 100 10-1

Wk0 vs 104 vs 117-B

*

***

sIgE/total IgE

100

103

102

101

10-1 10-2 10-3 10-4

Wk104

Wk117

10-1

Wk0

Wk104

Wk117

Wk0

Wk104

101 100 10-1 10-2

Wk117

10-4

Wk0

Wk104

Wk0 vs 104 vs 117-B

*** ***

102

N.S.

102 101 100 10-1

Wk117

*** *

***

101 100 10-1 10-2

***

Wk0 vs 104 vs 117-B

*** ***

103

***

102

***

***

101

102 101 100 10-1 10-2

***

10-3

Wk0 vs 104 vs 117-B

N.S.

Peanut specific IgG4 (ng/L)

**

N.S.

N.S.

100

10-2

10-2

10-1

101

10-2

*** ***

102

102

103

Ara h 2 IgE (kU/L)

Ara h 1 IgE (kU/L)

100

Wk0

102

103

***

***

Wk0 vs 104 vs 117-B

***

103

***

103

Wk117

***

Wk0 vs 104 vs 117-B

N.S.

101

104

*

10-2

Ara h 3 IgE (kU/L)

Wk117

102

Wk0 vs 104 vs 117-B

10-1

sIgG4/sIgE

Wk104

***

100

sIgG4/sIgE

***

10-3

103

101

***

***

Wk0 vs 104 vs 117-A

Peanut specific IgG4 (ng/L)

sIgE/total IgE

101

***

103

10-2

101

102

Wk0 vs 104 vs 117-B

102

Wk0 vs 104 vs 117-A

**

103

Wk0

***

sIgG4 wk0 vs 114 vs 117-A

**

104

Peanut specific IgE (kU/L)

103

N.S.

***

Wk0 vs 104 vs 117-A

N.S.

Ara h 3 IgE (kU/L)

102

Wk0 vs 104 vs 117-A

Total IgE (kU/L)

N.S.

***

Wk 0 vs 104 vs 117-A

***

103

Ara h 2 IgE (kU/L)

***

103

100

B

***

104

Ara h 1 IgE (kU/L)

Peanut specific IgE (kU/L)

Wk0 vs 104 vs 117-A

Total IgE (kU/L)

Peanut 0

A

Peanut 300

Figure 2

100 10-1 10-2 10-3

Wk0

Wk104

Wk117

10-4

Wk0

Wk104

Wk117

r=0.4358 r =p<0.0001 0.4358

P < 0.0001

103

102 101

101 100 10-1

100 50

100

150

50

100

150

101 100 10-1

50

100

WK0

r = 0.3894

102

P < 0.0001

100 10-1 10-2 10-3

50

100

150

10-4 0

200

50

high PF AUC %CD63high %CD63 PE AUC

P = 0.0001

101

100

100

r

sIgE/total IgE

Total IgE

102 101

50

100

high PF AUC %CD63 %CD63high PE AUC

150

100

150

Week 117

102

P < 0.0001

10-3

high PF AUC high %CD63 %CD63 PE AUC

50

100

150

r = 0.3144

100

50

100

150

high PF AUC %CD63high %CD63 PE AUC r=-0.2961

Week 117

r=0.06672 P=0.5515

rp=0.0069 = _0.2961

102

P = 0.55

P = 0.007

101

102 101 100 10-1 10-2 0

200

101

10-2 0

150

r = 0.0667

Week 117

10-2

150

10-1

103

P < 0.0001

100

P =r=0.3144 0.004 p=0.004

high PF AUC %CD63high %CD63 PE AUC

r=0.5314 p<0.0001

100

50

high %CD63 %CD63high PF PE AUC AUC

100

r = 0.5314

50

10-2

10-4 0

200

r=0.4766 rp<0.0001 = 0.4766

Week 117

10-2 0

150

10-1

10-4 0

100

101

high high %CD63 AUC %CD63 PEPF AUC

100

P = 0.68

103

100 0

50 Week 117

r=0.04236 = 0.0424 P=0.6819

104

50

10-1

10-2 0

150

10-1

10-3

102

101

high PF AUC %CD63 %CD63high PE AUC Week 117

10-1

103

P < 0.0001

10-1

100 50

100

r = 0.4491

Ara h 2 IgE

Ara h 1 IgE

102

Pp=0.0011 = 0.001

high PF AUC %CD63high %CD63 PE AUC

102

103

10-1 0

101

r=0.4491 p<0.0001

Week 117

103

r=0.4131

r p=0.0001 = 0.4131

200

100

10-2 0

200

150

r r=-0.2947 = _0.2947

101

P = 0.12

high PF AUC %CD63high %CD63 PE AUC

Week 117

104

150

Peanut specific IgG4

Week 117

Peanut specific IgE

B

100

100

Wk0

r=0.141 p=0.1225

Ara h 3 IgE

101 0

50

high PF AUC %CD63high %CD63 PE AUC

sIgG4/sIgE

103

10-2 0

200

r = 0.1417

WK0

102

r=0.3894 p<0.0001

Peanut specific IgG4

P p=0.0243 = 0.02

150

sIgG4/sIgE

101

sIgE/total IgE

Total IgE

WK0

r =r=0.2055 0.2055

104

100

high PF AUC %CD63high %CD63 PE AUC

high PF AUC %CD63high %CD63 PE AUC

high PF AUC %CD63 %CD63high PE AUC

101

10-1

10-3 0

200

r=0.3668 P < 0.0001 p<0.0001

102

10-2

10-2 0

200

r = 0.3668

Wk0

103

102

102

103

10-1 0

Week 0

r=0.4813 p<0.0001

P < 0.0001

Wk0

r=0.4033 P
103

Ara h 1 IgE

Peanut specific IgE

104

r = 0.4813

r = 0.4033

Wk0

Ara h 3 IgE

A

WK0

Ara h 2 IgE

Figure 3

100 10-1 10-2 10-3

50

100

high PF AUC high %CD63 %CD63 PE AUC

150

10-4 0

50

100

high PF AUC %CD63 %CD63high PE AUC

150

Figure 4 Failure

Success

A

B P=0.04

P=0.0004

P=0.001

P=0.03

P=0.06

P=0.0006

Peanut 0

P=0.006

P=0.02

P=0.049

P=0.0006

P=0.67

P=0.05

Peanut 300

C

D P=0.17

P=0.32

P=0.05

Peanut 0

P=0.09

P=0.93

P=0.44

P=0.19

P=0.50

Peanut 300

Figure 5 Peanut 0

100 10-1

102 101 100 10-1

P=0.25

103

102

100

101

B

10-2 10-3 10-4

101 100

100 10-1

P=0.002

102 101 100 10-1 10-2

Failure Peanut 0

Peanut 0

103

P=0.0006

101

102 101 100

P=0.01

103

100 10-1 10-2

10-1

10-3

10-2

102 101 100 10-1

Peanut 300

Peanut 300

P=0.03

103

10-2

10-1

P=0.02

103

102 101 100 10-1

P=0.06

102 101 100

Failure 10-1

Success

10-2

10-2

Peanut 300

Peanut 300

101

10-1 10-2 10-3 10-4

Peanut 300

103

Peanut 300

P=0.27

101

102 101 100 10-1 10-2

sIgG4/sIgE

102

100

P=0.04

Peanut specific IgG4 (ng/L)

103

P=0.80

sIgE/total IgE

Total IgE (kU/L)

104

Success

Peanut 300

P=0.05

102

101

Peanut 300

Ara h 1 IgE (kU/L)

Peanut specific IgE (kU/L)

P=0.002

10-1

Peanut 300

103

102

Peanut 0

sIgE/total IgE

Total IgE (kU/L)

104

103

10-2

10-2 Peanut 0

P<0.0001

sIgG4/sIgE

101

103

Ara h 3 IgE (kU/L)

102

P<0.0001

Ara h 3 IgE (kU/L)

103

Ara h 2 IgE (kU/L)

P<0.0001

Peanut specific IgG4 (ng/L)

103

Peanut 0

Peanut 0

Peanut 0

Ara h 1 IgE (kU/L)

Peanut specific IgE (kU/L)

Peanut 0

Ara h 2 IgE (kU/L)

A

100 10-1 10-2 10-3

P>0.9999

Figure 6 A

B

% CD63high PE AUC

200 150 100 50 0

C ITT

LR Peanut 0

IR HR

Peanut 300 Placebo 0

20

40

60

80

100

% P<0.0001 P=0.003

E 200

P<0.0001

CTD (mg)

D

P=0.047 CTD-wk0 P=0.04 P=0.60

150 100 50 0

LR

IR

HR

Week 0

10-2

P<0.0001 P<0.0001 P=0.01

102 101 100 10-1

103

101 100 10-1

102

10-1 10-2 10-3

sIgG4/sIgE

100 10-1 10-2

10-2

10-3

10-3

10-4

P=0.03 P=0.04

102 101 100

101 100 10-1

P=0.57

P=0.22

101 100 10-1

101

P=0.16 P=0.12

100 10-1 10-2 10-3 10-4

P=0.01

P=0.40

102

P=0.01

102

10-2

P=0.03 P=0.21

102

P=0.002

10-1

HR

P=0.006

P=0.48

102

100

IR

10-2

P=0.47

103

101

LR

10-1

103

101

Peanut specific IgG4 (ng/L)

101

P=0.0006 P=0.0002 P=0.08

102

103

P=0.006 P=0.42

10-2

10-1

P=0.008

102

P=0.64

101

10-1

100

104

P=0.002

100

P=0.48

100

P=0.06

10-4

101 P=0.51

101

10-2

Total IgE (kU/L)

P=0.59

103

102

P=0.002

101

sIgE/total IgE

Total IgE (kU/L)

P=0.10

103

103

P=0.02

102

P=0.11

102

P=0.04

10-2

10-2 104

P=0.0003

P=0.05 P=0.10

Ara h 1 IgE (kU/L)

10-1

103

Ara h 3 IgE (kU/L)

10-1

100

104

sIgE/total IgE

100

101

Peanut specific IgE (kU/L)

101

102

Ara h 2 IgE (kU/L)

102

103 Ara h 2 IgE (kU/L)

Ara h 1 IgE (kU/L)

103

P=0.004

P=0.01

P<0.0001

103 P=0.002 P=0.04

P=0.003 P=0.009

Week 117

B

101

sIgG4/sIgE

P<0.0001

104

Ara h 3 IgE (kU/L)

Peanut specific IgE (kU/L)

A

Peanut specific IgG4 (ng/L)

Figure 7

100 10-1 10-2 10-3

P=0.43

P=0.48

Figure E1

Week 0 Peanut 0

A

B

Peanut 300

C

Placebo

Figure E2

A

C

B ***

*

***

***

***

Peanut 0

***

***

*

***

**

***

***

***

Peanut 300

***

*

*

*

*

**

Placebo

Figure E3

A

P<0.0001 P=0.89

P<0.0001

B

P=0.07 P=0.47

P=0.009

Peanut 0 Peanut 300 Placebo

Week 104

Week 117

Figure E4

Failure

A

Success

B *

*

**

** **

**

*

Peanut 0 (Week 117)

Peanut 0 (Week 0)

C

D

** *

Peanut 300 (Week 0)

** *

Peanut 300 (Week 117)

Tsai, Mukai et al Supplementary material

Online Repository Material Sustained Successful Peanut Oral Immunotherapy Associated with Low Basophil Activation and Peanut-Specific IgE Mindy Tsai, DMSc1,2*, Kaori Mukai, PhD1,2*, R. Sharon Chinthrajah, MD1,2, Kari C. Nadeau, MD, PhD1,2, Stephen J. Galli, MD1,2,3,

1. Department of Pathology; 2. Sean N. Parker Center for Allergy and Asthma Research; 3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA * These authors contributed equally to this work

Correspondence to: Stephen J. Galli, MD Stanford University, 269 Campus Drive, Stanford, CA 94305-5101, USA. E-mail: [email protected] Phone: 650-736-0062 Fax: 540-736-0073

Funding: This work was supported by the National Institutes of Health [grant number U19 AI104209].

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Tsai, Mukai et al Supplementary material

Table E1. Plasma immunoglobulin levels in the Placebo arm. Peanut specific IgE (kU/L) Wk 0

175.90 + 63.61

P=0.002 vs. Wk 104 P=0.84 vs. Wk 117

Wk 104

113.99 + 37.02

P=0.01 vs. Wk 117

Wk 117

182.26 + 70.28

Wk 0

57.20 + 18.46

P=0.005 vs. Wk 104 P=0.85 vs. Wk 117

Wk 104 Wk 117

37.22 + 9.64 50.00 + 15.27

P=0.049 vs. Wk 117

Wk 0

74.14 + 27.93

P=0.001 vs. Wk 104 P=0.84 vs. Wk 117

Wk 104

43.66 + 13.91

P=0.03 vs. Wk 117

Wk 117

76.45 + 26.69

Wk 0

13.44 + 5.34

P=0.16 vs. Wk 104 P=0.56 vs. Wk 117

Wk 104

12.79 + 5.26

P=0.04 vs. Wk 117

Wk 117

12.97 + 5.23

Wk 0

884.32 + 247.71

P=0.35 vs. Wk 104 P=0.43 vs. Wk 117

Wk 104

723.73 + 137.18

P=0.59 vs. Wk 117

Wk 117

801.00 + 168.98

Ara h 1 IgE (kU/L)

Ara h 2 IgE (kU/L)

Ara h 3 IgE (kU/L)

Total IgE (kU/L)

Wk 104

sIgE/total IgE P=0.004 vs. Wk 104 0.15 + 0.03 P=0.15 vs. Wk 117 P=0.007 vs. Wk 117 0.12 + 0.02

Wk 117

0.14 + 0.03

Wk 0

Wk 104

Peanut specific IgG4 (ng/mL) P=0.0001 vs. Wk 104 1.41 + 0.46 P=0.04 vs. Wk 117 P=0.0002 vs. Wk 117 0.78 + 0.28

Wk 117

1.24 + 0.47

Wk 0

Wk 104

sIgG4/sIgE P=0.11 vs. Wk 104 0.18 + 0.12 P=0.46 vs. Wk 117 P=0.99 vs. Wk 117 0.11 + 0.05

Wk 117

0.14 + 0.06

Wk 0

P values were determined by Wilcoxon matched-pairs signed rank test. Data are mean + SEM. 2

Tsai, Mukai et al Supplementary material high

Table E2. Comparisons of %CD63 PE AUC and peanut-specific immunoglobulins between treatment failure and success in basophil intermediate and high responders before (Wk 0) and after (Wk 117) OIT. Peanut 0+ Peanut 300 high %CD63 PE AUC Wk 0 Failure 69.98+6.61 P=0.18 Success 56.52+5.99 Wk 117 Failure 24.89+3.26 *** P<0.0001 Success 8.90+1.61 Wk117/Wk 0 Failure 0.40+0.07 *** P<0.0001 Success 0.16+0.03 Wk 0 Wk 117 Wk117/Wk 0

Wk 0 Wk 117 Wk117/Wk 0

Peanut specific IgE Failure 255.55+41.58 Success 96.53+38.16 Failure 74.78+15.27 Success 30.71+10.23 Failure 0.36+0.05 Success 0.42+0.07 Ara h 1 IgE Failure 66.20+11.39 Success 19.86+6.28 Failure 18.42+3.87 Success 8.36+3.03

Failure 0.35+0.07 Success 0.63+0.09 Ara h 2 IgE Wk 0 Failure 131.88+23.47 Success 49.41+23.01 Wk 117 Failure 39.38+8.00 Success 13.04+5.06 Wk117/Wk 0 Failure 0.34+0.04 Success 0.41+0.05 Ara h 3 IgE Wk 0 Failure 16.55+4.44 Success 5.86+1.67 Wk 117 Failure 4.32+0.90 Success 1.96+0.64 Wk117/Wk 0 Failure 0.51+0.12 Success 0.67+0.07 Peanut specific IgG4 Wk 0 Failure 1.84+0.31 Success 2.04+0.83 Wk 117 Failure 29.53+4.90 Success 18.66+4.76 Wk117/Wk 0 Failure 25.64+5.03 Success 25.94+6.86

*** P<0.0001 *** P=0.0003 P= 0.86

*** P<0.0001 ** P=0.001 ** P=0.009 *** P<0.0001 *** P<0.0001 P=0.62

** P=0.005 ** P=0.005 * P=0.02 P=0.06 ** P=0.007 P=0.42

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Tsai, Mukai et al Supplementary material “Success” is defined as tolerant to DBPCFCs to 4 g peanut protein at week 117. P values are determined by Mann-Whitney test. Data are mean + SEM. * P < 0.05, ** P < 0.01, **P < 0.001

FIGURE LEGENDS FIG E1. Baseline basophil responsiveness to peanut, anti-IgE and IL-3 in Peanut 0, Peanut 300 and Placebo groups. A. Peanut-induced %CD63high basophils represented by area under the curve (AUC) as a function of the peanut extract (PE) protein dose (0-1000 ng/mL) in Peanut 0, Peanut 300 and Placebo arms. B. Anti-IgE (1 µg/mL)-induced %CD63high basophils in Peanut 0, Peanut 300 and Placebo arms. C. Anti-IgE (1 µg/mL), IL-3 (2 ng/mL) and peanut (0-1000 ng/mL)-induced CD203c in Peanut 0, Peanut 300 and Placebo arms. Data from participants who exited the study before week 104 were excluded. In A & B, Whiskers represent min to max, boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. Individual values are shown as circles. In C, Whiskers represent 10 to 90 percentile. Lower and higher 10% of all values are plotted individually. Boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. MFI, Mean fluorescence intensity.

FIG E2. Peanut OIT reduces basophil CD203c expression in responses to anti-IgE, IL-3 and peanut over time. A. Peanut 0. B. Peanut 300. C. Placebo. Data from participants who exited the study before week 104 were excluded. PE, peanut extract protein (0-1000 ng/mL). MFI, Mean fluorescence intensity. Whiskers represent 10 to 90 percentile. Lower and higher 10% of all values are plotted individually. Boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. P values were determined by mixed-effects analysis with the Geisser-Greenhouse correction. * P<0.05, **P<0.01, ***P<0.001.

FIG E3. Comparisons of peanut-induced basophil activation between the 3 study arms at weeks 104 and 117. A. Peanut induced %CD63high PE AUC after desensitization at week 104. B. Peanut induced %CD63high PE AUC at week 117, that was 13 weeks after active peanut OIT followed by either peanut withdrawal (Peanut 0) or 300 mg peanut maintenance (Peanut 300). Whiskers represent min to max, boxes 4

Tsai, Mukai et al Supplementary material

extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. Individual values are shown as circles. P values were determined by MannWhitney test.

FIG E4. Anti-IgE-, IL-3- and Peanut-induced ∆CD203c in basophils by week 117 treatment outcome. A. Treatment failure vs. success at week 0 in the Peanut 0 arm. B. Treatment failure vs. success at week 117 in the Peanut 0 arm. C. Treatment failure vs. success at week 0 in the Peanut 300 arm. D. Treatment failure vs. success at week 117 in the Peanut 300 arm. Data from participants who exited the study before week 104 were excluded. Whiskers represent 10 to 90 percentile. Lower and higher 10% of all values are plotted individually. Boxes extend from the 25th to 75th percentiles. The lines in the middle of the boxes are medians. P values were determined by MannWhitney test. * P<0.05, **P<0.01, ***P<0.001. “Success” is tolerance in DBPCFCs to 4 g peanut protein at week 117.

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